Polymerase variants and uses thereof

ABSTRACT

Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. patent application Ser. No. 15/151,364(filed May 10, 2016), which is a Continuation-in-Part of U.S.application Ser. No. 15/012,317 (filed Feb. 1, 2016 and issued as U.S.Pat. No. 10,308,918 B2 on Jun. 4, 2019), which claims priority to U.S.Provisional Patent Application 62/161,571 (filed on May 14, 2015) andU.S. Provisional Patent Application 62/202,895 (filed on Aug. 9, 2015).Each of the foregoing patents and patent applications is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Nov. 25, 2019, is namedP32651 US6_SL_ST25 and is 18,009 bytes in size.

TECHNICAL FIELD

Provided herein, among other things, are modified DNA polymerasescontaining amino acid alterations based on mutations identified indirected evolution experiments designed to select enzymes that arebetter suited for applications in recombinant DNA technologies.

BACKGROUND

DNA polymerases are a family of enzymes that use single-stranded DNA asa template to synthesize the complementary DNA strand. In particular,DNA polymerases can add free nucleotides to the 3′ end of anewly-forming strand resulting in elongation of the new strand in a 5′to 3′ direction. Most DNA polymerases are multifunctional proteins thatpossess both polymerizing and exonucleolytic activities. For example,many DNA polymerases have 3′→5′ exonuclease activity. These polymerasescan recognize an incorrectly incorporated nucleotide and the 3′→5′exonuclease activity of the enzyme allows the incorrect nucleotide to beexcised (this activity is known as proofreading). Following nucleotideexcision, the polymerase can re-insert the correct nucleotide andreplication can continue. Many DNA polymerases also have 5′→3′exonuclease activity.

Polymerases have found use in recombinant DNA applications, includingnanopore sequencing. However, a DNA strand moves rapidly at the rate of1 μs to 5 μs per base through the nanopore. This makes recordingdifficult and prone to background noise, failing in obtainingsingle-nucleotide resolution. Therefore, the use of detectable tags onnucleotides may be used in the sequencing of a DNA strand or fragmentthereof. Thus, there is a not only a need to control the rate of DNAbeing sequenced but also provide polymerases that have improvedproperties (relative to the wild-type enzyme) such as incorporation ofmodified nucleotides, e.g., polyphosphate nucleotides with or withouttags.

BRIEF SUMMARY OF THE INVENTION

The present invention provides modified DNA polymerases (e.g., mutants)based on directed evolution experiments designed to select mutationsthat confer advantageous phenotypes under conditions used in industrialor research applications, e.g., catalyzing incorporation of modifiedpolyphosphate nucleotides, e.g., tagged nucleotides, under high saltconcentrations.

In an aspect there is a variant polymerase comprising at least onealteration at a position corresponding to of H223, N224, Y225, H227,I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417,E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546,A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623,D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652,K655, W656, D657, V658, H660, F662, and L690 of SEQ ID NO:2 (Pol6 (withHis tag)).

In one embodiment there is provided a modified DNA polymerase having aDNA polymerase activity comprising an amino acid sequence having atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%sequence identity to the amino acid sequence as set forth in SEQ ID NO:1or 2.

In a second embodiment there is provided a modified DNA polymerasehaving a DNA polymerase activity comprising an amino acid sequencehaving at least 70%, at least 75%, at least 80%, at least 90% or atleast 95% sequence identity to the amino acid sequence as set forth inSEQ ID NO:1 or 2 having one or more amino acid substitutions selectedfrom the group consisting of H223, N224, Y225, H227, I295, Y342, T343,I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478,K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550,N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629,R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657,V658, H660, F662, and L690 and combinations thereof. In a furtherembodiment, the one or more amino acid substitutions are selected fromH223A, N224Y/L/Q/M/R/K, Y225L/T/l/F/A/M, H227P/E/F/Y, I295W/F/M/E,Y342L/F, T343N/F, I357G/L/Q/H/W/M/A/E/Y/P, S360G/E/Y, L361 M/W/V/F,I363V/A/R/M/W, S365Q/W/M/A/G, S366A/L, Y367L/E/M/P/N/F, P368G, D417P,E475D, Y476V, F478L, K518Q, H527W/R/L/Y/T/M, T529M/F,M531H/Y/A/K/R/W/T/L/V/G, N535L/Y/M/K/I/R/W/Q, G539Y/F, P542E/S/G,N545K/D/S/L/R/Q/W, Q546W/F, A547M/Y/W/F/V/S, L549Q/Y/H/G/R/K,I550A/W/T/G/F/S, N552L/M/S/T, G553S/T/E/Q/K/R/M, F558P/T, A596S,G603T/A/L, A610T/E, V615A/T, Y622A/M, C623G/S/Y/F, D624F/K,I628Y/V/F/L/M, Y629W/H/M/K, R632L/C, N635D, M641 L/Y, A643L, I644H/M/Y,T647G/A/E/K/S/Y, I648K/R/V/N/T/L, T651Y/F/M, I652Q/G/S/N/F/T/A/L/E/K/M,K655G/F/E/N/Q/M/A, W656E, D657R/P/A/E, V658L, H660A/Y, F662I/L, L690Mand combinations thereof. The modified DNA polymerase having one or moreamino acid substitutions has an altered characteristic selected fromenzyme activity, fidelity, processivity, elongation rate, sequencingaccuracy, long continuous read capability, stability, and solubilityrelative to the parental polymerase. In an embodiment, the alteredcharacteristic is enzyme activity. In an embodiment, the alteredcharacteristic is fidelity. In an embodiment, the altered characteristicis processivity. In an embodiment, the altered characteristic iselongation rate. In an embodiment, the altered characteristic isstability. In an embodiment, the altered characteristic is solubility.In one embodiment, the altered characteristic is an ability to bindand/or incorporate polyphosphate nucleotides, e.g., a tetraphosphate,pentaphosphate, hexaphosphate, heptaphosphate or octophosphatenucleotide.

In a third embodiment, there is provided a modified DNA polymerasehaving an altered characteristic selected from enzyme activity,fidelity, processivity, elongation rate, stability, or solubility, whencompared to SEQ ID NO:1 or 2. In an embodiment, the alteredcharacteristic is enzyme activity. In an embodiment, the alteredcharacteristic is fidelity. In an embodiment, the altered characteristicis processivity. In an embodiment, the altered characteristic iselongation rate. In an embodiment, the altered characteristic isstability. In an embodiment, the altered characteristic is solubility.

In a fourth embodiment, there is provided a modified DNA polymerasehaving a DNA polymerase activity comprising an amino acid sequencehaving at least 70%, at least 75%, at least 80%, at least 90% or atleast 95% sequence identity to the amino acid sequence as set forth inSEQ ID NO:1, which amino acid sequence includes one or more amino acidsubstitutions, such substitutions being selected from the groupconsisting of H223A, N224Y/L/Q/M/R/K, Y225L/T/l/F/A, H227P/E/F/Y,I295W/F/M/E, Y342L/F, T343N/F, I357G/L/Q/H/W/M/A/E/Y/P, S360G/E/Y,L361M/W/V/F, I363V/A/R/M/W, S365Q/W/M/A/G, S366A/L, Y367L/E/M/P/N/F,P368G, D417P, E475D, Y476V, F478L, K518Q, H527W/R/L/Y/T/M, T529M/F,M531H/Y/A/K/R/W/T/L/V/G, N535L/Y/M/K/I/R/W/Q, G539Y/F, P542E/S/G,N545K/D/S/L/R/Q/W, Q546W/F, A547M/Y/W/F/V/S, L549Q/Y/H/G/R/K,I550A/W/T/G/F/S, N552L/M/S/T, G553S/T/E/Q/K/R/M, F558P/T, A596S,G603T/A/L, A610T/E, V615A/T, Y622A/M, C623G/S/Y/F, D624F/K,I628Y/V/F/L/M, Y629W/H/M/K, R632L/C, N635D, M641 L/Y, A643L, I644H/M/Y,T647G/A/E/K/S/Y, I648K/R/V/N/T/L, T651Y/F/M, I652Q/G/S/N/F/T/A/L/E/K/M,K655G/F/E/N/Q/M/A, W656E, D657R/P/A/E, V658L, H660A/Y, F662I/L, L690Mand combinations thereof, wherein the one or more amino acidsubstitutions alter enzyme activity, fidelity, processivity, elongationrate, sequencing accuracy, long continuous read capability, stability,or solubility relative to the parental polymerase. In an embodiment, thealtered characteristic is enzyme activity. In an embodiment, the alteredcharacteristic is fidelity. In an embodiment, the altered characteristicis processivity. In an embodiment, the altered characteristic iselongation rate. In an embodiment, the altered characteristic isstability. In an embodiment, the altered characteristic is solubility.In one embodiment, the altered characteristic is an ability to bindand/or incorporate polyphosphate nucleotides, e.g., a tetraphosphate,pentaphosphate, hexaphosphate, heptaphosphate or octophosphatenucleotide.

In an embodiment, the variant polymerase having altered enzyme activityas compared to SEQ ID NO:1 or 2 is selected from

-   -   a. H223A;    -   b. N224Y/L/Q/M/R/K;    -   c. Y225L/I/T/F/A/M;    -   d. H227P/E/F/Y;    -   e. I295F/E/M/W;    -   f. Y342L/F;    -   g. T343N/F;    -   h. I357G/L/Q/H/W/M/A/E/Y/P;    -   i. S360G/E/Y;    -   j. L361M/W/V/F;    -   k. I363V/A/R/M/W;    -   l. S365Q/W/M/A/G;    -   m. S366A/L;    -   n. Y367L/E/M/P/N/F;    -   o. P368G;    -   p. D417P;    -   q. E475D;    -   r. Y476V;    -   s. F478L;    -   t. K518Q;    -   u. H527W/R/L/Y/T/M;    -   v. T529M/F;    -   w. M531H/Y/A/K/R/W/T/L/V/G;    -   x. N535L/Y/M/K/I/R/W/O;    -   y. P542E/S/G;    -   z. N545D/K/S/L/R/Q/W;    -   aa. Q546W/F;    -   bb. A547F/M/W/Y/V/S;    -   cc. L549H/Y/Q/G/R/K;    -   dd. I550A/W;    -   ee. I550T/G/F/S;    -   ff. N552L/M/T;    -   gg. G553S/T/E/Q/K/R/M;    -   hh. F558P/T;    -   ii. A596S;    -   jj. G603T/A/L;    -   kk. A610T/E;    -   ll. V615A/T;    -   mm. Y622A/M;    -   nn. C623G/S/Y/A/F;    -   oo. D624F/K;    -   pp. I628Y/V/F/L/M;    -   qq. Y629W/H/M/K;    -   rr. R632L/C;    -   ss. N635D;    -   tt. M641L/Y;    -   uu. A643L;    -   vv. I644H/M/Y;    -   ww. T647G/A/E/K/S/Y;    -   xx. I648K/R/V/N/T/L;    -   yy. T651Y/F/M;    -   zz. I652Q/G/S/N/F/T/A/L/E/K/M;    -   aaa. K655G/F/E/N/Q/M/A;    -   bbb. W656E;    -   ccc. D657R/P/A/E;    -   ddd. V658L;    -   eee. H660A/Y;    -   fff. F662I/L;    -   ggg. L690M;    -   hhh. S366A+N535L;    -   iii. T651Y+N535L;    -   jjj. Y342L+E475D+F478L;    -   kkk. T343N+D417P+K518Q;    -   lll. N535L+N545K+T651Y;    -   mmm. I363V+E475D+Y476V;    -   nnn. S366L+G553S+F558P;    -   ooo. S366A+N535L+A547M;    -   ppp. S366A+P542E+N545K;    -   qqq. S366A+P542E+I652Q;    -   rrr. S366A+N535L+T529M;    -   sss. S366A+N535L+I652Q;    -   ttt. S366A+N535L+N545K;    -   uuu. T651Y+P542E+N545K;    -   vvv. I651Y+P542E+Q546 W;    -   www. I651Y+P542E+S366A;    -   xxx. I651Y+N535L+N545K;    -   yyy. S366A+N535I+I652Q;    -   zzz. I651Y+S366A+A547F;    -   aaaa. T647G+A547F+Y225T;    -   bbbb. A547F+A610T+S366A;    -   cccc. A547F+A610T+Y225I;    -   dddd. S366A+T647G+A547F;    -   eeee. T529M+S366A+A547F;    -   ffff. T647E+S366A+A547F;    -   gggg. T529M+T647G+A547F;    -   hhhh. N545K+S366A+A547F;    -   iiii. T647G+A547F+T529M;    -   jjjj. T529M+A610T+A547F;    -   kkkk. M641Y+T529M+A547F;    -   llll. I647G+C623G+A547F;    -   mmmm. A610T+I295W+T651Y;    -   nnnn. V615A+M531Y+T647G;    -   oooo. S366L+F478L+A596S+L690M;    -   pppp. H223A+G553S+A643L+F662I;    -   qqqq. N535L+N545K+T651Y+T529M;    -   rrrr. N535L+N545K+T651Y+N635D;    -   ssss. N535L+N545K+T651Y+I652Q;    -   tttt. S366A+N535L+I652Q+T529M;    -   uuuu. S366A+S365A+P368G+G603T;    -   vvvv. N535L+N545K+T651Y+T647G;    -   wwww. S366A+N535L+I652Q+A547Y;    -   xxxx. S366A+N535L+A547M+T647G;    -   yyyy. T529M+S366A+A547F+N545K;    -   zzzz. T529M+S366A+A547F+N545R;    -   aaaaa. T529M+S366A+A547F+N552L;    -   bbbbb. T529M+S366A+A547F+Y629 W;    -   ccccc. N535I+N545K+T651Y+T529M;    -   ddddd. N535I+N545K+T651Y+N635D;    -   eeeee. N535I+N545K+T651Y+I652Q;    -   fffff. N535L+N545K+T651Y+T647G+C623G;    -   ggggg. N535L+N545K+T651Y+T647G+I628Y;    -   hhhhh. S366A+N535L+A547M+T647G+S360G;    -   iiiii. N535I+N545K+T651Y+I652Q+Y225I;    -   jjjjj. N535L+N545K+T651Y+T647G+K655G;    -   kkkkk. N535L+N545K+T651Y+T647G+L549Q;    -   lllll. S366A+N535L+I652Q+A547Y+K655G;    -   mmmmm. T529M+S366A+A547F+N545L+Y629 W;    -   nnnnn. T529M+S366A+A547F+N545L+Y225L;    -   ooooo. T529M+S366A+A547F+N545L+Y225F;    -   ppppp. T529M+S366A+A547F+N545L+K655F;    -   qqqqq. T529M+S366A+A547F+N545L+N552L;    -   rrrrr. T529M+S366A+A547F+N545R+M531A;    -   sssss. T529M+S366A+A547F+N545R+G539Y;    -   ttttt. T529M+S366A+A547F+N545R+V658L;    -   uuuuu. T529M+S366A+A547F+N545L+Y225L+D657R;    -   vvvvv. T529M+S366A+A547F+N545L+Y225L+N552L;    -   wwwww. T529M+S366A+A547F+N545L+Y225L+I652G;    -   xxxxx. T529M+S366A+A547F+N545L+Y225L+I652Q;    -   yyyyy. T529M+S366A+A547F+N545L+Y225L+N552M    -   zzzzz. T529M+S366A+A547F+N545L+Y225L+D657R+N224R;    -   aaaaaa. T529M+S366A+A547F+N545L+Y225L+D657R+I628M;    -   bbbbbb. T529M+S366A+A547F+N545L+Y225L+D657R+K655A; and    -   cccccc. T529M+S366A+A547F+N545L+Y225L+D657R+Y629 W.        In an embodiment, the altered characteristic is enzyme activity.        In an embodiment, the altered characteristic is fidelity. In an        embodiment, the altered characteristic is processivity. In an        embodiment, the altered characteristic is elongation rate. In an        embodiment, the altered characteristic is stability. In an        embodiment, the altered characteristic is solubility. In one        embodiment, the altered characteristic is an ability to bind        and/or incorporate polyphosphate nucleotides, e.g., a        tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate or        octophosphate nucleotide.

In some embodiments, the variant polymerase having altered enzymeactivity, as compared to SEQ ID NO:2 having the N535I+N545K+T651Y+N635Dmutations, or SEQ ID NO:1 or 2 is selected from

-   -   a. A547F+A610T+Y225I;    -   b. Y225T+T647G+A547F;    -   c. S366A+T647G+A547F;    -   d. S366A+A547F+A610T;    -   e. T529M+S366A+A547F;    -   f. T529M+T647G+A547F;    -   g. T529M+A610T+A547F;    -   h. N545K+S366A+A547F;    -   i. N545K+T647G+A547F;    -   j. A610T+I295W+T651Y;    -   k. V615A+M531Y+T647G;    -   l. M641Y+T529M+A547F;    -   m. T647E+S366A+A547F;    -   n. T647G+A547F+T529M;    -   o. T647G+C623G+A547F; and    -   p. T651Y+S366A+A547F.

In some embodiments, the variant polymerase is selected from

-   -   a. N535L+N545K+T651Y;    -   b. S366A+N535L+I652Q;    -   c. S366A+T529M+N535L;    -   d. S366A+N535L+N545K;    -   e. S366A+N535L+A547M;    -   f. S366A+P542E+I652Q;    -   g. S366A+P542E+N545K;    -   h. S366A+P542E+T651Y;    -   i. P542E+N545K+T651Y;    -   j. P542E+Q546W+T651Y;    -   k. N535L+T651Y;    -   l. S366A+N535L;    -   m. N535L+N545K+T651Y+T529M;    -   n. N535L+N545K+T651Y+N635D;    -   o. N535L+N545K+T651Y+I652Q;    -   p. S366A+N535L+I652Q+T529M;    -   q. N535L+N545K+T651Y+T647G;    -   r. S366A+N535L+I652Q+A547Y;    -   s. S366A+N535L+A547M+T647G;    -   t. S366A+N535I+I652Q;    -   u. N535I+N545K+T651Y+T529M;    -   v. N535I+N545K+T651Y+N635D;    -   w. N535I+N545K+T651Y+I652Q;    -   x. N535L+N545K+T651Y+T647G+C623G;    -   y. N535L+N545K+T651Y+T647G+I628Y;    -   z. S366A+N535L+A547M+T647G+S360G;    -   aa. N535I+N545K+T651Y+I652Q+Y225I;    -   bb. N535L+N545K+T651Y+T647G+K655G;    -   cc. N535L+N545K+T651Y+T647G+L549Q;    -   dd. S366A+N535L+I652Q+A547Y+K655G;    -   ee. T647G+A547F+Y225T;    -   ff. A547F+A610T+S366A;    -   gg. A547F+A610T+Y225I;    -   hh. S366A+T647G+A547F;    -   ii. I651Y+S366A+A547F;    -   jj. T529M+S366A+A547F;    -   kk. T647E+S366A+A547F;    -   ll. T529M+T647G+A547F;    -   mm. N545K+S366A+A547F;    -   nn. T647G+A547F+T529M;    -   oo. N545K+T647G+A547F;    -   pp. T529M+A610T+A547F;    -   qq. M641Y+T529M+A547F;    -   rr. T647G+C623G+A547F;    -   ss. A610T+I295W+T651Y;    -   tt. V615A+M531Y+T647G;    -   uu. T529M+S366A+A547F+N545K;    -   vv. T529M+S366A+A547F+N545R;    -   ww. T529M+S366A+A547F+N552L;    -   xx. T529M+S366A+A547F+Y629 W;    -   yy. T529M+S366A+A547F+N545L+Y629 W;    -   zz. T529M+S366A+A547F+N545L+Y225L;    -   aaa. T529M+S366A+A547F+N545L+Y225F;    -   bbb. T529M+S366A+A547F+N545L+K655F;    -   ccc. T529M+S366A+A547F+N545L+N552L;    -   ddd. T529M+S366A+A547F+N545R+M531A;    -   eee. T529M+S366A+A547F+N545R+G539Y;    -   fff. T529M+S366A+A547F+N545R+V658L;    -   ggg. T529M+S366A+A547F+N545L+Y225L+D657R;    -   hhh. T529M+S366A+A547F+N545L+Y225L+N552L;    -   iii. T529M+S366A+A547F+N545L+Y225L+I652G;    -   jjj. T529M+S366A+A547F+N545L+Y225L+I652Q;    -   kkk. T529M+S366A+A547F+N545L+Y225L+N552M    -   lll. T529M+S366A+A547F+N545L+Y225L+D657R+N224R;    -   mmm. T529M+S366A+A547F+N545L+Y225L+D657R+I628M;    -   nnn. T529M+S366A+A547F+N545L+Y225L+D657R+K655A; and    -   ooo. T529M+S366A+A547F+N545L+Y225L+D657R+Y629 W.        In some embodiments, the variant polymerase having altered        enzyme activity as compared to SEQ ID NOs:1 or 2, or the        parental polymerase.

In some embodiments, the variant polymerase having altered enzymeactivity, as compared to SEQ ID NO:2 having the S366A+T529M+N545L+A547Fmutations, or SEQ ID NO:1 or 2, is selected from

-   -   a. Y225L/F/A/M;    -   b. M531A/G;    -   c. G539Y;    -   d. N552L/T;    -   e. Y629W/K;    -   f. K655F.        In an embodiment, the altered characteristic is enzyme activity.        In an embodiment, the altered characteristic is fidelity. In an        embodiment, the altered characteristic is processivity. In an        embodiment, the altered characteristic is elongation rate. In an        embodiment, the altered characteristic is stability. In an        embodiment, the altered characteristic is solubility. In one        embodiment, the altered characteristic is an ability to bind        and/or incorporate polyphosphate nucleotides, e.g., a        tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate or        octophosphate nucleotide.

In some embodiments, the parental polymerase is wild-type Pol6 (SEQ IDNO:1). In some embodiments, the parental polymerase is Pol6 comprising aHis-tag (SEQ ID NO:2). In some embodiments, the parental polymerase iscomprises the mutations S366A+T529M+A547F+N545L/R. In some embodiments,the parental polymerase may be SEQ ID NO:1 comprising one or moremutations. For example, S366A+T529M+A547F+N545R used S366A+T529M+A547Fas the parental polymerase then added N545R.

In some embodiments, the modified polymerase has a k_(chem) that isgreater than the parental polymerase. In some embodiments, the modifiedpolymerase has a k_(off) that is less than the parental polymerase. Insome embodiments, the modified polymerase has a k_(chem)/k_(off) (i.e.,a ratio) that is at least 1.5, 2.0 or 2.5 times greater than theparental polymerase.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary template used in the displacement assay.Reference is made to Example 3.

FIG. 2 shows a schematic of the k_(chem) assay used herein to measurethe rate of incorporation of polyphosphates. Reference is made toExample 6.

FIG. 3 is a summary of the fluorescence quenching based k_(off) assayused herein to measure kinetic properties of the variant polymerases.Reference is made to Example 4.

FIG. 4 is a depiction of the k_(off) assay based on fluorescencepolarization and an exemplary data trace. Reference is made to Example5.

FIG. 5 is a graph showing representative data from the displacementassay for a variant polymerase. Reference is made to Example 3.

FIG. 6 is a graph of representative data from fluorescence polarizationbased k_(off) assay for two variant polymerases. Reference is made toExample 5.

FIG. 7 is a trace of a static capture of tagged thymine nucleotide at100 mV by Pol6 (S366A+N535L+I652Q)-DNA complex coupled toalpha-hemolysin nanopore in 20 mM Hepes7.5, 300 mM NaCl, 3 mM CaCl2 and5 mM TCEP above and below the bilayer. The vertical axis is % openchannel current (normalized) and horizontal axis is time in seconds.Reference is made to Example 8.

FIG. 8 is graph of Dwell time vs current plot for a static captureexperiment at 100 mV with Pol6 (S366A+N535L+I652Q)-DNA complex coupledto alpha-hemolysin nanopore in 20 mM Hepes pH 7.5, 300 mM NaCl, 3 mMCaCl2 and 5 mM TCEP above and below the bilayer. The average dwell timeof each capture of dTNP-tagged nucleotide is 1.2 seconds. Reference ismade to Example 8.

FIG. 9 is a graph of representative data from a fluorescence quenchingbased k_(chem) assay (see FIG. 2) for a variant polymerase. Preformedbinary complex of polymerase and Fluorescein-DNA template is mixedrapidly with saturating concentration of dCnP-Alexa555 in the presenceof Mg²⁺ using a Kintek stopped flow device. Fluorescein fluorescence ismonitored over time. k_(chem) estimated from the rate limiting step is0.2 s-1. The x-axis is time (T) in seconds and the y-axis is relativefluorescence units (RFU).

FIG. 10 is a graph of representative data from a fluorescence quenchingbased k_(off) assay (see FIG. 3) for a variant polymerases. Preformedternary complex of polymerase, Fluorescein-DNA template anddCnP-Alexa555 was preincubated in the presence of Ca²⁺ and chased withexcess native dCTP. Fluorescein fluorescence was monitored over time.k_(off) measured from this is 0.028 s-1. The x-axis is time (T) inseconds and the y-axis is relative fluorescence units (RFU).

FIG. 11 is a picture of a gel showing the amplification products of arolling circle assay. The left and right end lanes are molecularladders. The lane second from the left is the zero time point. All otherlanes are the 40-minute time point for the various polymerase hits.Reference is made to Example 9.

FIG. 12 is a sequencing trace showing the changes in current thatprovides a record of the tagged nucleotides as they are incorporatedinto the growing DNA strand. Also shown is the template DNA sequence andthe called sequence of the nascent strand demonstrating >70% accuracy(SEQ ID NOS 6-8, respectively, in order of appearance). Reference ismade to Example 10.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Practitioners areparticularly directed to Sambrook et al., 1989, and Ausubel F M et al.,1993, for definitions and terms of the art. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. The termabout is used herein to mean plus or minus ten percent (10%) of a value.For example, “about 100” refers to any number between 90 and 110.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Definitions

Amino acid: As used herein, the term “amino acid,” in its broadestsense, refers to any compound and/or substance that can be incorporatedinto a polypeptide chain. In some embodiments, an amino acid has thegeneral structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acidis a naturally-occurring amino acid. In some embodiments, an amino acidis a synthetic amino acid; in some embodiments, an amino acid is aD-amino acid; in some embodiments, an amino acid is an L-amino acid.“Standard amino acid” refers to any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acid”refers to any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or obtained from anatural source. As used herein, “synthetic amino acid” encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and/or substitutions. Aminoacids, including carboxy- and/or amino-terminal amino acids in peptides,can be modified by methylation, amidation, acetylation, and/orsubstitution with other chemical without adversely affecting theiractivity. Amino acids may participate in a disulfide bond. The term“amino acid” is used interchangeably with “amino acid residue,” and mayrefer to a free amino acid and/or to an amino acid residue of a peptide.It will be apparent from the context in which the term is used whetherit refers to a free amino acid or a residue of a peptide. It should benoted that all amino acid residue sequences are represented herein byformulae whose left and right orientation is in the conventionaldirection of amino-terminus to carboxy-terminus.

Base Pair (bp): As used herein, base pair refers to a partnership ofadenine (A) with thymine (T), or of cytosine (C) with guanine (G) in adouble stranded DNA molecule.

Complementary: As used herein, the term “complementary” refers to thebroad concept of sequence complementarity between regions of twopolynucleotide strands or between two nucleotides through base-pairing.It is known that an adenine nucleotide is capable of forming specifichydrogen bonds (“base pairing”) with a nucleotide which is thymine oruracil. Similarly, it is known that a cytosine nucleotide is capable ofbase pairing with a guanine nucleotide.

DNA binding affinity: As used herein, the term “DNA-binding affinity”typically refers to the activity of a DNA polymerase in binding DNAnucleic acid. In some embodiments, DNA binding activity can be measuredin a two band-shift assay. See, e.g., Sambrook et al. (2001) MolecularCloning: A Laboratory Manual (3^(rd) ed., Cold Spring Harbor LaboratoryPress, N.Y.) at 9.63-9.75 (describing end-labeling of nucleic acids). Areaction mixture is prepared containing at least about 0.5 μg of thepolypeptide in about 10 μl of binding buffer (50 mM sodium phosphatebuffer (pH 8.0), 10% glycerol, 25 mM KCl, 25 mM MgCl₂). The reactionmixture is heated to 37° C. for 10 min. About 1×10⁴ to 5×10⁴ cpm (orabout 0.5-2 ng) of the labeled double-stranded nucleic acid is added tothe reaction mixture and incubated for an additional 10 min. Thereaction mixture is loaded onto a native polyacrylamide gel in 0.5×Tris-borate buffer. The reaction mixture is subjected to electrophoresisat room temperature. The gel is dried and subjected to autoradiographyusing standard methods. Any detectable decrease in the mobility of thelabeled double-stranded nucleic acid indicates formation of a bindingcomplex between the polypeptide and the double-stranded nucleic acid.Such nucleic acid binding activity may be quantified using standarddensitometric methods to measure the amount of radioactivity in thebinding complex relative to the total amount of radioactivity in theinitial reaction mixture. Other methods of measuring DNA bindingaffinity are known in the art (see, e.g., Kong et al. (1993) J. Biol.Chem. 268(3):1965-1975).

Elongation rate: As used herein, the term “elongation rate” refers tothe average rate at which a DNA polymerase extends a polymer chain. Asused herein, a high elongation rate refers to an elongation rate higherthan 2 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s). Asused in this application, the terms “elongation rate”, “extension rate”and “incorporation rate” are used inter-changeably.

Enzyme activity: As used herein, the term “enzyme activity” refers tothe specificity and efficiency of a DNA polymerase. Enzyme activity of aDNA polymerase is also referred to as “polymerase activity,” whichtypically refers to the activity of a DNA polymerase in catalyzing thetemplate-directed synthesis of a polynucleotide. Enzyme activity of apolymerase can be measured using various techniques and methods known inthe art. For example, serial dilutions of polymerase can be prepared indilution buffer (e.g., 20 mM Tris.Cl, pH 8.0, 50 mM KCl, 0.5% NP 40, and0.5% Tween-20). For each dilution, 5 μl can be removed and added to 45μl of a reaction mixture containing 25 mM TAPS (pH 9.25), 50 mM KCl, 2mM MgCl₂, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.1 mM dCTP, 12.5 μgactivated DNA, 100 μM [α-³²P]dCTP (0.05 μCi/nmol) and sterile deionizedwater. The reaction mixtures can be incubated at 37° C. (or 74° C. forthermostable DNA polymerases) for 10 minutes and then stopped byimmediately cooling the reaction to 4° C. and adding 10 μl of ice-cold60 mM EDTA. A 25 μl aliquot can be removed from each reaction mixture.Unincorporated radioactively labeled dCTP can be removed from eachaliquot by gel filtration (Centri-Sep, Princeton Separations, Adelphia,N.J.). The column eluate can be mixed with scintillation fluid (1 ml).Radioactivity in the column eluate is quantified with a scintillationcounter to determine the amount of product synthesized by thepolymerase. One unit of polymerase activity can be defined as the amountof polymerase necessary to synthesize 10 nmole of product in 30 minutes(Lawyer et al. (1989) J. Biol. Chem. 264:6427-647). Other methods ofmeasuring polymerase activity are known in the art (see, e.g. Sambrooket al. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., ColdSpring Harbor Laboratory Press, N.Y.)).

Purified: As used herein, “purified” means that a molecule is present ina sample at a concentration of at least 90% by weight, or at least 95%by weight, or at least 98% by weight of the sample in which it iscontained.

Isolated: An “isolated” molecule is a nucleic acid molecule that isseparated from at least one other molecule with which it is ordinarilyassociated, for example, in its natural environment. An isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the nucleic acid molecule, but the nucleic acidmolecule is present extrachromasomally or at a chromosomal location thatis different from its natural chromosomal location.

% homology: The term “% homology” is used interchangeably herein withthe term “% identity” herein and refers to the level of nucleic acid oramino acid sequence identity between the nucleic acid sequence thatencodes any one of the inventive polypeptides or the inventivepolypeptide's amino acid sequence, when aligned using a sequencealignment program.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence, e.g., the coding sequence for anyone of the inventive polypeptides, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet. See also, Altschul, et al., 1990 andAltschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402, 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 13.0.7, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

Modified DNA polymerase: As used herein, the term “modified DNApolymerase” refers to a DNA polymerase originated from another (i.e.,parental) DNA polymerase and contains one or more amino acid alterations(e.g., amino acid substitution, deletion, or insertion) compared to theparental DNA polymerase. In some embodiments, a modified DNA polymerasesof the invention is originated or modified from a naturally-occurring orwild-type DNA polymerase. In some embodiments, a modified DNA polymeraseof the invention is originated or modified from a recombinant orengineered DNA polymerase including, but not limited to, chimeric DNApolymerase, fusion DNA polymerase or another modified DNA polymerase.Typically, a modified DNA polymerase has at least one changed phenotypecompared to the parental polymerase.

Mutation: As used herein, the term “mutation” refers to a changeintroduced into a parental sequence, including, but not limited to,substitutions, insertions, deletions (including truncations). Theconsequences of a mutation include, but are not limited to, the creationof a new character, property, function, phenotype or trait not found inthe protein encoded by the parental sequence.

Mutant: As used herein, the term “mutant” refers to a modified proteinwhich displays altered characteristics when compared to the parentalprotein. The terms “variant” and “mutant” are used interchangeablyherein.

Wild-type: As used herein, the term “wild-type” refers to a gene or geneproduct which has the characteristics of that gene or gene product whenisolated from a naturally-occurring source.

Fidelity: As used herein, the term “fidelity” refers to either theaccuracy of DNA polymerization by template-dependent DNA polymerase orthe measured difference in k_(off) of the correct nucleotide vsincorrect nucleotide binding to the template DNA. The fidelity of a DNApolymerase is typically measured by the error rate (the frequency ofincorporating an inaccurate nucleotide, i.e., a nucleotide that is notincorporated at a template-dependent manner). The accuracy or fidelityof DNA polymerization is maintained by both the polymerase activity andthe 3′-5′ exonuclease activity of a DNA polymerase. The term “highfidelity” refers to an error rate less than 4.45×10⁻⁶ (e.g., less than4.0×10⁻⁶, 3.5×10⁻⁶, 3.0×10⁻⁶, 2.5×10⁻⁶, 2.0×10⁻⁶, 1.5×10⁻⁶, 1.0×10⁻⁶,0.5×10⁻⁶) mutations/nt/doubling. The fidelity or error rate of a DNApolymerase may be measured using assays known to the art. For example,the error rates of DNA polymerases can be tested as described herein oras described in Johnson, et al., Biochim Biophys Acta. 2010 May;1804(5): 1041-1048.

Nanopore: The term “nanopore,” as used herein, generally refers to apore, channel or passage formed or otherwise provided in a membrane. Amembrane may be an organic membrane, such as a lipid bilayer, or asynthetic membrane, such as a membrane formed of a polymeric material.The membrane may be a polymeric material. The nanopore may be disposedadjacent or in proximity to a sensing circuit or an electrode coupled toa sensing circuit, such as, for example, a complementary metal-oxidesemiconductor (CMOS) or field effect transistor (FET) circuit. In someexamples, a nanopore has a characteristic width or diameter on the orderof 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins.Alpha-hemolysin, MspA are examples of a protein nanopore.

Nucleotide: As used herein, a monomeric unit of DNA or RNA consisting ofa sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclicbase. The base is linked to the sugar moiety via the glycosidic carbon(1′ carbon of the pentose) and that combination of base and sugar is anucleoside. When the nucleoside contains a phosphate group bonded to the3′ or 5′ position of the pentose it is referred to as a nucleotide. Asequence of operatively linked nucleotides is typically referred toherein as a “base sequence” or “nucleotide sequence,” and is representedherein by a formula whose left to right orientation is in theconventional direction of 5′-terminus to 3′-terminus. As used herein, a“modified nucleotide” refers to a polyphosphate, e.g., 3, 4, 5, 6, 7 or8 phosphates, nucleotide.

Oligonucleotide or Polynucleotide: As used herein, the term“oligonucleotide” is defined as a molecule including two or moredeoxyribonucleotides and/or ribonucleotides, preferably more than three.Its exact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe derived synthetically or by cloning. As used herein, the term“polynucleotide” refers to a polymer molecule composed of nucleotidemonomers covalently bonded in a chain. DNA (deoxyribonucleic acid) andRNA (ribonucleic acid) are examples of polynucleotides.

Polymerase: As used herein, a “polymerase” refers to an enzyme thatcatalyzes the polymerization of nucleotide (i.e., the polymeraseactivity). Generally, the enzyme will initiate synthesis at the 3′-endof the primer annealed to a polynucleotide template sequence, and willproceed toward the 5′ end of the template strand. A “DNA polymerase”catalyzes the polymerization of deoxynucleotides.

Primer: As used herein, the term “primer” refers to an oligonucleotide,whether occurring naturally or produced synthetically, which is capableof acting as a point of initiation of nucleic acid synthesis when placedunder conditions in which synthesis of a primer extension product whichis complementary to a nucleic acid strand is induced, e.g., in thepresence of four different nucleotide triphosphates and thermostableenzyme in an appropriate buffer (“buffer” includes pH, ionic strength,cofactors, etc.) and at a suitable temperature. The primer is preferablysingle-stranded for maximum efficiency in amplification, but mayalternatively be double-stranded. If double-stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the thermostableenzyme. The exact lengths of the primers will depend on many factors,including temperature, source of primer and use of the method. Forexample, depending on the complexity of the target sequence, theoligonucleotide primer typically contains 15-25 nucleotides, although itmay contain more or few nucleotides. Short primer molecules generallyrequire colder temperatures to form sufficiently stable hybrid complexeswith template.

Processivity: As used herein, “processivity” refers to the ability of apolymerase to remain attached to the template and perform multiplemodification reactions. “Modification reactions” include but are notlimited to polymerization, and exonucleolytic cleavage. In someembodiments, “processivity” refers to the ability of a DNA polymerase toperform a sequence of polymerization steps without interveningdissociation of the enzyme from the growing DNA chains. Typically,“processivity” of a DNA polymerase is measured by the length ofnucleotides (for example 20 nts, 300 nts, 0.5-1 kb, or more) that arepolymerized or modified without intervening dissociation of the DNApolymerase from the growing DNA chain. “Processivity” can depend on thenature of the polymerase, the sequence of a DNA template, and reactionconditions, for example, salt concentration, temperature or the presenceof specific proteins. As used herein, the term “high processivity”refers to a processivity higher than 20 nts (e.g., higher than 40 nts,60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts,220 nts, 240 nts, 260 nts, 280 nts, 300 nts, 320 nts, 340 nts, 360 nts,380 nts, 400 nts, or higher) per association/disassociation with thetemplate. Processivity can be measured according the methods definedherein and in WO 01/92501 A1 (M J Bioworks, Inc., Improved Nucleic AcidModifying Enzymes, published 6 Dec. 2001).

Synthesis: As used herein, the term “synthesis” refers to any in vitromethod for making new strand of polynucleotide or elongating existingpolynucleotide (i.e., DNA or RNA) in a template dependent mannerSynthesis, according to the invention, includes amplification, whichincreases the number of copies of a polynucleotide template sequencewith the use of a polymerase. Polynucleotide synthesis (e.g.,amplification) results in the incorporation of nucleotides into apolynucleotide (i.e., a primer), thereby forming a new polynucleotidemolecule complementary to the polynucleotide template. The formedpolynucleotide molecule and its template can be used as templates tosynthesize additional polynucleotide molecules. “DNA synthesis,” as usedherein, includes, but is not limited to, PCR, the labeling ofpolynucleotide (i.e., for probes and oligonucleotide primers),polynucleotide sequencing.

Template DNA molecule: As used herein, the term “template DNA molecule”refers to a strand of a nucleic acid from which a complementary nucleicacid strand is synthesized by a DNA polymerase, for example, in a primerextension reaction.

Template-dependent manner: As used herein, the term “template-dependentmanner” refers to a process that involves the template dependentextension of a primer molecule (e.g., DNA synthesis by DNA polymerase).The term “template-dependent manner” typically refers to polynucleotidesynthesis of RNA or DNA wherein the sequence of the newly synthesizedstrand of polynucleotide is dictated by the well-known rules ofcomplementary base pairing (see, for example, Watson, J. D. et al., In:Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., MenloPark, Calif. (1987)).

Tag: As used herein, the term “tag” refers to a detectable moiety thatmay be atoms or molecules, or a collection of atoms or molecules. A tagmay provide an optical, electrochemical, magnetic, or electrostatic(e.g., inductive, capacitive) signature, which signature may be detectedwith the aid of a nanopore.

Tagged Nucleotide: As used herein, the term “tagged nucleotide” refersto a nucleotide or modified nucleotide that has a tag attached. The tagmay be attached covalently to the sugar, the phosphate (orpolyphosphate) or base. The tag may be on the terminal phosphate.

Vector: As used herein, the term “vector” refers to a nucleic acidconstruct designed for transfer between different host cells. An“expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are commerciallyavailable. Selection of appropriate expression vectors is within theknowledge of those having skill in the art.

The polymerase variants provided for herein are useful in the chip-basedpolynucleotide sequencing as described in WO2013/188841 (GeniaTechnologies, Inc., Chip Set-Up and High-Accuracy Nucleic AcidSequencing, published 19 Dec. 2013).

Desired characteristics of a polymerase that finds use in sequencing DNAare:

-   -   a. Slow k_(off) (for modified nucleotide)    -   b. Fast k_(on) (for modified nucleotide)    -   c. High fidelity    -   d. Low exonuclease activity    -   e. DNA strand displacement    -   f. Faster k_(chem) (for modified nucleotide substrates)    -   g. Increased stability    -   h. Processivity    -   i. Salt tolerance    -   j. Compatible with attachment to nanopore    -   k. Ability to incorporate a polyphosphates having 4, 5, 6, 7 or        8 phosphates, e.g., quadraphosphate, pentaphosphate,        hexaphosphate, heptaphosphate or octophosphate nucleotide    -   l. Sequencing accuracy    -   m. Long read lengths, i.e., long continuous reads.        Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used.

For ease of reference, polymerase variants of the application aredescribed by use of the following nomenclature:

Original amino acid(s): position(s): substituted amino acid(s).According to this nomenclature, for instance the substitution of serineby an alanine in position 242 is shown as:

-   -   Ser242Ala or S242A

Multiple mutations are separated by plus signs, i.e.:

-   -   Ala30Asp+Glu34Ser or A30N+E34S        representing mutations in positions 30 and 34 substituting        alanine and glutamic acid for asparagine and serine,        respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as: A30N/E or A30N or A30E.

Unless otherwise stated, the number of the residues corresponds to theresidue numbering of SEQ ID NO:2.

Site-Directed Mutagenesis of Polymerase

Clostridium phage phiCPV4 wild type sequences are provided herein (SEQID NO:3, nucleic acid coding region plus a His-tag; SEQ ID NO:1, proteincoding region) and available elsewhere (National Center forBioinformatics or GenBank Accession Numbers AFH27113).

Point mutations may be introduced using QuikChange Lightning 2 kit(Stategene/Agilent) following manufacturer's instructions.

Primers can be ordered from commercial companies, e.g., IDT DNA.

Nanopore Assembly and Insertion

The methods described herein can use a nanopore having a polymeraseattached to the nanopore. In some cases, it is desirable to have one andonly one polymerase per nanopore (e.g., so that only one nucleic acidmolecule is sequenced at each nanopore). However, many nanopores,including, e.g., alpha-hemolysin (aHL), can be multimeric proteinshaving a plurality of subunits (e.g., 7 subunits for aHL). The subunitscan be identical copies of the same polypeptide. Provided herein aremultimeric proteins (e.g., nanopores) having a defined ratio of modifiedsubunits (e.g., a-HL variants) to un-modified subunits (e.g., a-HL).Also provided herein are methods for producing multimeric proteins(e.g., nanopores) having a defined ratio of modified subunits toun-modified subunits.

With reference to FIG. 27 of WO2014/074727 (Genia Technologies, Inc.), amethod for assembling a protein having a plurality of subunits comprisesproviding a plurality of first subunits 2705 and providing a pluralityof second subunits 2710, where the second subunits are modified whencompared with the first subunits. In some cases, the first subunits arewild-type (e.g., purified from native sources or producedrecombinantly). The second subunits can be modified in any suitable way.In some cases, the second subunits have a protein (e.g., a polymerase)attached (e.g., as a fusion protein).

The modified subunits can comprise a chemically reactive moiety (e.g.,an azide or an alkyne group suitable for forming a linkage). In somecases, the method further comprises performing a reaction (e.g., a Clickchemistry cycloaddition) to attach an entity (e.g., a polymerase) to thechemically reactive moiety.

The method can further comprise contacting the first subunits with thesecond subunits 2715 in a first ratio to form a plurality of proteins2720 having the first subunits and the second subunits. For example, onepart modified aHL subunits having a reactive group suitable forattaching a polymerase can be mixed with six parts wild-type aHLsubunits (i.e., with the first ratio being 1:6). The plurality ofproteins can have a plurality of ratios of the first subunits to thesecond subunits. For example, the mixed subunits can form severalnanopores having a distribution of stoichiometries of modified toun-modified subunits (e.g., 1:6, 2:5, 3:4).

In some cases, the proteins are formed by simply mixing the subunits. Inthe case of aHL nanopores for example, a detergent (e.g., deoxycholicacid) can trigger the aHL monomer to adopt the pore conformation. Thenanopores can also be formed using a lipid (e.g.,1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC)) and moderatetemperature (e.g., less than about 100° C.). In some cases, mixing DPhPCwith a buffer solution creates large multi-lamellar vesicles (LMV), andadding aHL subunits to this solution and incubating the mixture at 40°C. for 30 minutes results in pore formation.

If two different types of subunits are used (e.g., the natural wild typeprotein and a second aHL monomer which can contain a single pointmutation), the resulting proteins can have a mixed stoichiometry (e.g.,of the wild type and mutant proteins). The stoichiometry of theseproteins can follow a formula which is dependent upon the ratio of theconcentrations of the two proteins used in the pore forming reaction.This formula is as follows:100 P _(m)=100[n!/m!(n−m)!]·f _(mut) ^(m) ·f _(wt) ^(n˜m),where

-   -   P_(m)=probability of a pore having m number of mutant subunits    -   n=total number of subunits (e.g., 7 for aHL)    -   m=number of “mutant” subunits    -   f_(mut)=fraction or ratio of mutant subunits mixed together    -   f_(wt)=fraction or ratio of wild-type subunits mixed together

The method can further comprise fractionating the plurality of proteinsto enrich proteins that have a second ratio of the first subunits to thesecond subunits 2725. For example, nanopore proteins can be isolatedthat have one and only one modified subunit (e.g., a second ratio of1:6). However, any second ratio is suitable. A distribution of secondratios can also be fractionated such as enriching proteins that haveeither one or two modified subunits. The total number of subunitsforming the protein is not always 7 (e.g., a different nanopore can beused or an alpha-hemolysin nanopore can form having six subunits) asdepicted in FIG. 27 of WO2014/074727. In some cases, proteins havingonly one modified subunit are enriched. In such cases, the second ratiois 1 second subunit per (n−1) first subunits where n is the number ofsubunits comprising the protein.

The first ratio can be the same as the second ratio, however this is notrequired. In some cases, proteins having mutated monomers can form lessefficiently than those not having mutated subunits. If this is the case,the first ratio can be greater than the second ratio (e.g., if a secondratio of 1 mutated to 6 non-mutated subunits are desired in a nanopore,forming a suitable number of 1:6 proteins may require mixing thesubunits at a ratio greater than 1:6).

Proteins having different second ratios of subunits can behavedifferently (e.g., have different retention times) in a separation. Insome cases, the proteins are fractionated using chromatography, such asion exchange chromatography or affinity chromatography. Since the firstand second subunits can be identical apart from the modification, thenumber of modifications on the protein can serve as a basis forseparation. In some cases, either the first or second subunits have apurification tag (e.g., in addition to the modification) to allow orimprove the efficiency of the fractionation. In some cases, apoly-histidine tag (His-tag), a streptavidin tag (Strep-tag), or otherpeptide tag is used. In some instances, the first and second subunitseach comprise different tags and the fractionation step fractionates onthe basis of each tag. In the case of a His-tag, a charge is created onthe tag at low pH (Histidine residues become positively charged belowthe pKa of the side chain). With a significant difference in charge onone of the aHL molecules compared to the others, ion exchangechromatography can be used to separate the oligomers which have 0, 1, 2,3, 4, 5, 6, or 7 of the “charge-tagged” aHL subunits. In principle, thischarge tag can be a string of any amino acids which carry a uniformcharge. FIG. 28 and FIG. 29 show examples of fractionation of nanoporesbased on a His-tag. FIG. 28 shows a plot of ultraviolet absorbance at280 nanometers, ultraviolet absorbance at 260 nanometers, andconductivity. The peaks correspond to nanopores with various ratios ofmodified and unmodified subunits. FIG. 29 of WO2014/074727 showsfractionation of aHL nanopores and mutants thereof using both His-tagand Strep-tags.

In some cases, an entity (e.g., a polymerase) is attached to the proteinfollowing fractionation. The protein can be a nanopore and the entitycan be a polymerase. In some instances, the method further comprisesinserting the proteins having the second ratio subunits into a bilayer.

In some situations, a nanopore can comprise a plurality of subunits. Apolymerase can be attached to one of the subunits and at least one andless than all of the subunits comprise a first purification tag. In someexamples, the nanopore is alpha-hemolysin or a variant thereof. In someinstances, all of the subunits comprise a first purification tag or asecond purification tag. The first purification tag can be apoly-histidine tag (e.g., on the subunit having the polymeraseattached).

Polymerase Attached to Nanopore

In some cases, a polymerase (e.g., DNA polymerase) is attached to and/oris located in proximity to the nanopore. The polymerase can be attachedto the nanopore before or after the nanopore is incorporated into themembrane. In some instances, the nanopore and polymerase are a fusionprotein (i.e., single polypeptide chain).

The polymerase can be attached to the nanopore in any suitable way. Insome cases, the polymerase is attached to the nanopore (e.g., hemolysin)protein monomer and then the full nanopore heptamer is assembled (e.g.,in a ratio of one monomer with an attached polymerase to 6 nanopore(e.g., hemolysin) monomers without an attached polymerase). The nanoporeheptamer can then be inserted into the membrane.

Another method for attaching a polymerase to a nanopore involvesattaching a linker molecule to a hemolysin monomer or mutating ahemolysin monomer to have an attachment site and then assembling thefull nanopore heptamer (e.g., at a ratio of one monomer with linkerand/or attachment site to 6 hemolysin monomers with no linker and/orattachment site). A polymerase can then be attached to the attachmentsite or attachment linker (e.g., in bulk, before inserting into themembrane). The polymerase can also be attached to the attachment site orattachment linker after the (e.g., heptamer) nanopore is formed in themembrane. In some cases, a plurality of nanopore-polymerase pairs areinserted into a plurality of membranes (e.g., disposed over the wellsand/or electrodes) of the biochip. In some instances, the attachment ofthe polymerase to the nanopore complex occurs on the biochip above eachelectrode.

The polymerase can be attached to the nanopore with any suitablechemistry (e.g., covalent bond and/or linker). In some cases, thepolymerase is attached to the nanopore with molecular staples. In someinstances, molecular staples comprise three amino acid sequences(denoted linkers A, B and C). Linker A can extend from a hemolysinmonomer, Linker B can extend from the polymerase, and Linker C then canbind Linkers A and B (e.g., by wrapping around both Linkers A and B) andthus the polymerase to the nanopore. Linker C can also be constructed tobe part of Linker A or Linker B, thus reducing the number of linkermolecules.

In some instances, the polymerase is linked to the nanopore usingSolulink™ chemistry. Solulink™ can be a reaction between HyNic(6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB(4-formylbenzoate, an aromatic aldehyde). In some instances, thepolymerase is linked to the nanopore using Click chemistry (availablefrom LifeTechnologies for example). In some cases, zinc finger mutationsare introduced into the hemolysin molecule and then a molecule is used(e.g., a DNA intermediate molecule) to link the polymerase to the zincfinger sites on the hemolysin.

Other linkers that may find use in attaching the polymerase to ananopore are direct genetic linkage (e.g., (GGGGS)₁₋₃ (SEQ ID NO:4)amino acid linker), transglutaminase mediated linking (e.g., RSKLG(SEQID NO:5)), sortase mediated linking, and chemical linking throughcysteine modifications. Specific linkers contemplated as useful hereinare (GGGGS)₁₋₃ (SEQ ID NO:4), K-tag (RSKLG(SEQ ID NO:5)) on N-terminus,ΔTEV site (12-25), ΔTEV site+N-terminus of SpyCatcher (12-49).

Apparatus Set-Up

The nanopore may be formed or otherwise embedded in a membrane disposedadjacent to a sensing electrode of a sensing circuit, such as anintegrated circuit. The integrated circuit may be an applicationspecific integrated circuit (ASIC). In some examples, the integratedcircuit is a field effect transistor or a complementary metal-oxidesemiconductor (CMOS). The sensing circuit may be situated in a chip orother device having the nanopore, or off of the chip or device, such asin an off-chip configuration. The semiconductor can be anysemiconductor, including, without limitation, Group IV (e.g., silicon)and Group III-V semiconductors (e.g., gallium arsenide). See, forexample, WO 2013/123450, for the apparatus and device set-up for sensinga nucleotide or tag.

Pore based sensors (e.g., biochips) can be used forelectro-interrogation of single molecules. A pore based sensor caninclude a nanopore of the present disclosure formed in a membrane thatis disposed adjacent or in proximity to a sensing electrode. The sensorcan include a counter electrode. The membrane includes a trans side(i.e., side facing the sensing electrode) and a cis side (i.e., sidefacing the counter electrode).

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds).

EXAMPLES

The present invention is described in further explained in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention.

Example 1 Directed Mutagenesis

This example illustrates the introduction of a mutation into a pol6polymerase at a desired position.

DNA encoding the His-tagged wild-type pol6 was purchased from acommercial source (DNA 2.0, Menlo Park, Calif.). The sequence wasverified by sequencing.

For the mutant screen, we expressed the polymerase as is (N-terHis-Pol6). In order to test the pol hits on the chip, we engineered in aSpyCatcher domain in N-ter or C-ter of Pol6.

Rational positions to impact Pol6-nucleotide binding were identifiedbased on homology modeling of known crystal structures.

For the primary screen, each of the rational positions were mutated intoGly, Ala, Leu, Glu, Gln, Lys, His, Tyr, Pro, Trp, Thr or Met using theQ5 mutagenesis protocol.

The primers for each mutagenesis reaction was designed using the NEBbase changer protocol and ordered in 96-well plate format from IDT.

The forward and reverse primers were 5′ phosphorylated in highthroughput (HTP) format using the T4 polynucleotidekinase (PNK)purchased from NEB. A typical 25-μl reaction contained 15 μl of primerat 10 μM, 5 μl of 5× reaction buffer (from NEB), 1.25 μl PNK enzyme,3.75 μl water. The reaction was performed at 37° C. for 30 min and theenzyme heat inactivated at 65° C. for 20 min.

PCR mutagenesis was performed using Q5 DNA polymerase from NEB. Atypical 25 μl reaction contained 5 μl of Q5 buffer, 5 μl of GC enhancer,0.5 μl of 10 mM dNTPs, 1.25 μl of 10 μM phosphorylated mutagenesisprimers forward and reverse, 0.25 μl Q5 polymerase and 1 μl of 5 ng/mlwild type Pol6 template, i.e., His-Pol6, and 10.75 μl H₂O.

Once PCR is complete, 0.5 μl of Dpn1 was added to 25 μl PCR mix andincubated at 37° C. for 1 hr.

Add 2.5 μl of Dpn1 treated PCR product with 2.5 μl of Blunt/TA ligasemaster mix. Incubate at room temperature for 1 hr.

Add 1 μl of ligation mix to 20 ul of 96-well BL21 DE3 cells (EMDMillipore) and incubate on ice for 5 min.

Heat shock at 42° C. for exactly 30 sec using the PCR device and placeon ice for 2 min.

Add 80 μl of SOC and incubate at 37° C. incubator for 1 hr withoutshaking.

Add 100 μl of SOC or ultra pure water and plate them in 48-well LB-agarplates with 50-100 μg/ml kanamycin.

Example 2 Expression and Purification

The following example details how the pol6 variants were expressed andpurified using a high throughput method.

DNA encoding the variants in the pD441 vector (expression plasmid) wastransformed into competent E. coli and glycerol stocks made. Startingfrom a tiny pick of the glycerol stock, grow 1 ml starter culture in LBwith 0.2% Glucose and 100 μg/ml Kanamycin for approximately 8 hrs.Transfer 25 μl of log phase starter culture into 1 ml of expressionmedia (Terrific Broth (TB) autoinduction media supplemented with 0.2%glucose, 50 mM Potassium Phosphate, 5 mM MgCl2 and 100 μg/ml Kanamycin)in 96-deep well plates. The plates were incubated with shaking at250-300 rpm for 36-40 hrs at 28° C.

Cells were then harvested via centrifugation at 3200×g for 30 minutes at4° C. The media was decanted off and the cell pellet resuspended in 200μl pre-chilled lysis buffer (20 mM Potassium Phosphate pH 7.5, 100 mMNaCl, 0.5% Tween20, 5 mM TCEP, 10 mM Imidazole, 1 mM PMSF, 1× BugBuster, 100 μg/ml Lysozyme and protease inhibitors) and incubate at roomtemperature for 20 min with mild agitation. Then add 20 μl from a 10×stock to a final concentration of 100 μg/ml DNase, 5 mM MgCl2, 100 μg/mlRNase I and incubate in on ice for 5-10 min to produce a lysate.Supplement the lysate with 200 μl of 1M Potassium Phosphate, pH 7.5(Final concentration will be about 0.5M Potassium phosphate in 400 μllysate) and filter through Pall filter plates (Part #5053, 3 micronfilters) via centrifugation at approximately 1500 rpm at 4C for 10minutes. The clarified lysates were then applied to equilibrated 96-wellHis-Pur Cobalt plates (Pierce Part #90095) and bind for 15-30 min.

The flow through (FT) was collected by centrifugation at 500×G for 3min. The FT was then washed 3 times with 400 ul of wash buffer 1 (0.5MPotassium Phosphate pH 7.5, 1M NaCl 5 mM TCEP, 20 mM Imidazole+0.5%Tween20). The FT was then washed twice in 400 ul wash buffer 2 (50 mMTris pH 7.4, 200 mM KCl, 5 mM TCEP, 0.5% Tween20, 20 mM Imidazole).

The Pol6 was eluted using 200 μl elution buffer (50 mM Tris Ph7.4, 200mM KCl, 5 mM TCEP, 0.5% Tween20, 300 mM Imidazole, 25% Glycerol) andcollected after 1-2 min incubation. Reapply eluate to the same His-Purplate2-3 times to get concentrated Pol6 in elute. The purifiedpolymerase is >95% pure as evaluated by SDS-PAGE. The proteinconcentration is ˜3 uM (0.35 mg/ml) with a 260/280 ratio of 0.6 asevaluated by Nanodrop.

Polymerase activity is checked by Fluorescence displacement assay (seeExample 3).

Example 3 Determination of Activity

This example provides methods of determining the activity of the variantpolymerases.

Displacement Assay Protocol

This assay characterizes the mutant polymerase's ability to incorporatepolyphosphate nucleotides into a DNA strand as well as its ability tounwind and displace double-stranded DNA.

Stock Reagents are as Follows:

Low Salt Reagent A Reagent B Reagent Concentration Concentration KCl 21.4 mM 20 mM Bicine 7.9 26.75 mM 25 mM EDTA 0.284 mM N/A Triton X-1000.0535% 0.05% DTT  5.35 mM  5 mM BSA 26.75 μg/ml 25 μg/ml DNA FRET   71nM N/A Template MgSO4 N/A 20 mM N/A = not applicable

High Salt Reagent A Reagent B Reagent Concentration Concentration NaCl  75 mM 300 or 150 mM HEPES 7.5 32.6 mM 25 mM EDTA  0.3 mM N/A TritonX-100 0.065% 0.05% TCEP  6.5 mM 5 mM BSA 32.6 μg/ml 25 μg/ml DNA FRET  87 nM N/A Template MgCl N/A 20 mM N/A = not applicable

For Screening Single and Double Mutants:

Using Reagent A as diluent, make 4 different nucleotide conditions at1.42×:

Nucleotide [1.42X] [Final] 1 dTnP-NH2 28.4 μM 20 μM dATP 21.3 μM 15 μMdCTP 21.3 μM 15 μM dGTP 21.3 μM 15 μM 2 dTnP-NH2 2.84 μM  2 μM dATP 21.3μM 15 μM dCTP 21.3 μM 15 μM dGTP 21.3 μM 15 μM 3 dTnP-NH2   0 μM  0 μMdATP 21.3 μM 15 μM dCTP 21.3 μM 15 μM dGTP 21.3 μM 15 μM 4 dTnP-NH2 0 0dATP 0 0 dCTP 0 0 dGTP 0 0

For Screening Triple Mutants:

Using Reagent A as diluent, make 4 different nucleotide conditions at1.42×:

Nucleotide [1.42X] [Final] 1 dTnP-NH2 28.4 μM 20 μM dAnP-NH2 28.4 μM 20μM dCnP-NH2 28.4 μM 20 μM dGnP-NH2 28.4 μM 20 μM 2 dTnP-NH2 1.42 μM  1μM dAnP-NH2 1.42 μM  1 μM dCnP-NH2 1.42 μM  1 μM dGnP-NH2 1.42 μM  1 μM3 dTnP-NH2   0 μM  0 μM dATP 21.3 μM 15 μM dCTP 21.3 μM 15 μM dGTP 21.3μM 15 μM 4 dTnP-NH2 0 0 dAnP-NH2 0 0 dCnP-NH2 0 0 dGnP-NH2 0 0dNnP is a polyphosphate nucleotide where N is the nucleotide (i.e., A,T, C or G) and nP is 4-8 phosphates

Nucleotide condition 1 tests for activity at high concentration of thehexaphosphate.

Nucleotide condition 2 tests for activity at low concentration of thehexaphosphate.

Nucleotide condition 3 tests for misincorporation rate (i.e., fidelity).If a mutant polymerase shows significant activity with only 3 of the 4necessary nucleotides, then we conclude that it does not discriminatebetween correct or incorrect nucleotides while extending a DNA strand.

Nucleotide condition 4 tests for exonuclease activity. If a polymeraseshows significant activity with no nucleotides present, then we concludethe polymerase is exhibiting exonuclease activity.

To each reaction well in a 96 well half-area transparent plate, add:

-   -   23 μl Reagent A/nucleotide mix    -   2 μl polymerase (1-10 μM)

Shake at 800 RPM on plate shaker for ˜10 min.

Add 5 μl 1.4 M NaCl to each well to bring the NaCl concentration up to300 mM or 5 μl 525 mM NaCl to each well to bring the NaCl concentrationup to 150 mM.

Incubate for 30 minutes.

In BMG LABTECH plate reader, inject 10 μl reagent B and readfluorescence signal for 2 to 10 min.

Representative data from the displacement assay for a variant polymeraseare shown in FIG. 5. The activity of polymerase was measured using thedisplacement assay in the presence of A. 20 μM dTnP+15 μM dA,C,G3P (redsquares; ▪), 5 μM dTnP+15 μM dA,C,G3P (blue diamonds; ♦), C. 15 μMdA,C,G3P (green triangles; ▴), or D. in the absence of nucleotides(purple X's). A and B show that a mutant variant is able to incorporateand extend along a DNA template with a polyphosphate nucleotide. C showsthat the variant has not lost its fidelity and is not misincorporatingrandom nucleotides in the absence of a T nucleotide. D. shows that thesignal generated is not a result of the polymerase exonuclease activityin the absence of all nucleotides. All four curves are representative ofa single variant tested across 4 different assay plates as part ofpolymerase screen.

Example 4 Determination of K_(OFF)

The following stopped flow assay was used to determine the k_(off) rateof the variant polymerases.

For reagent A, polymerase is bound to a fluorescein labeled DNAtemplate-primer with a Cy3 (or Alexa555)-linked polyphosphate nucleotidein the presence of a non-catalytic divalent metal like Ca2+. This formsa FRET pair, fluorescein being the donor fluorophore and Cy3 being theacceptor fluorophore. Reagent B contains the chase nucleotide. Forpurposes of this assay, the first nucleotide to be incorporated into thetemplate/primer is Cytosine.

Reagent A (75 mM NaCl, 25 mM HEPES (pH 7.5), 2 mM CaCl2), 250 nMFluorescein-Template/Primer, 20 uM dCnP-Cy3, and >250 nM Polymerase) wasfreshly prepared by mixing the components ensuring that the polymeraseis added last. Allow the polymerase to incubate in Reagent A for 10minutes.

Reagent B (75 mM NaCl, 25 mM HEPES (pH 7.5), 2 mM CaCl2), and 200 uMdCTP) was prepared.

When reagent A and B are mixed, dCTP competes with dCnP-Cy3 forassociation, an increase in fluorescence is observed given the dCTPconcentration is in excess. The assay can be performed with either astop flow device (Kintek Corp) or a fluorescent plate reader. Theincrease in fluorescence versus time was fit to a first order or secondorder exponential to provide the kinetic constant k_(off) for thatparticular polymerase.

The purification yields and k_(off)s for selected variants are presentedin Table 1.

TABLE 1 Purification yields and k_(off)s for select Pol6 variants Yieldfrom 2.5 ml k_(off) (s⁻¹) prep (μg) Good k_(off) hit and moderately goodactivity at 20 uM S366A + N535L + A547M 0.0039 77.58 Hits from 20mod(Good activity at 20 uM hexaphosphate nucleotide & very low or noactivity at 0 uM) T651Y + P542E + N545K 0.0562 5.16 T651Y + P542E +Q546W 0.0201 64.58 S366A + P542E + N545K 0.0295 125.24 Hits from 20MSR(Good activity at 20 uM, activity at 0 uM) S366A + P542E + I652Q 0.0440196.63 T651Y + P542E + S366A 0.0583 10.11 Hits from 1MSR/1Mod and alsoshow moderately good activity at 20 uM S366A + N535L + N545K 0.010360.25 T651Y + N535L + N545K 0.0273 210.66 S366A + N535L + T529M 0.0327153.09 S366A + N535L + I652Q 0.0097 161.55 Double mutant hit T651Y +N535L 0.0312 281.84 S366A + N535L 0.0134 666.95 FP = Fluorescentpolarization

See FIG. 3 for a schematic representation of the assay and a graph of anexemplary reaction. See FIG. 10 for representative data.

Example 5 Determination of K_(OFF)

This example provides an alternative method using fluorescencepolarization for determining the k_(off).

An assay buffer comprising 25 mM Tris pH7.0, 75 mM KCl, 0.01%Triton-X100, 1×BSA (100 ug/ml), 0.5 mM EDTA, 2 mM CaCl₂), 2 mM DTT, wasused to prepare an assay master mix containing 250 nM hairpinfluorescein-labeled DNA template and 250 nM dC6P-C6-Cy3 taggednucleotide. Fifty five microliters of the master mix were added to eachof the wells of a black 96-well costar plate; and. 20 μl of polymerasemutants, which had been purified from 1 ml cultures, were added in ahigh throughput (HTP) format. The plate was shaken on a plate shaker for1 minute to allow for the formation of homogenous ternary complexes ofpolymerase-DNA template-nucleotide. The plate was placed in a BMGpolarstar plate reader (BMG LABTECH Inc., North Carolina) and targetmillipolarization was adjusted to 200 mP and 10% to have a gain around2000. The excitation filter was set to 485 nM and the emission filterwas set to 590-20 nM. The injector was primed with 1 ml of 1 mM dCTPchaser nucleotide solution. Data was collected with minimum 30 flashesper well per interval and 60 sec total read time for the start. Theflashes were increased to 50 or higher and longer read times taken forthe hit mutants that showed slow dissociation. Data collection beganwith the injection of 25 μl of 1 mM dCTP.

See FIG. 4 for a schematic representation of the assay and a graph of anexemplary reaction.

See FIG. 6 for representative data from fluorescence polarization basedk_(off) assay for two variant polymerases (S366A+N535L+I652Q (B6) andS366A+P542E+I652Q (C6)). mP is millipolarization. Preformed ternarycomplex of polymerase-DNA template-dCnP-Alexa555 is chased with nativedCTP and polarization dCnP-Alexa555 was monitored over time.

Example 6 Determination of K_(CHEM)

This example provides a FRET based assay for determining the k_(chem)for variant polymerases.

For reagent A, polymerase is bound to fluorescein labeled DNAtemplate-primer. Reagent B contains Cy3 (or Alexa555)-linkedpolyphosphate nucleotide in the presence of a catalytic divalent metallike Mg²⁺. For purposes of this protocol, the first nucleotide to beincorporated into the template/primer is Cytosine.

Reagent A (75 mM NaCl, 25 mM HEPES (pH 7.5), 250 nMFluorescein-Template/Primer, >250 nM Polymerase) was prepared. Thepolymerase was allowed to incubate in Reagent A for 10 min.

Reagent B (75 mM NaCl, 25 mM HEPES (pH 7.5), 10 mM MgCl₂, and 20 uMdCnP-Cy3) was prepared.

When Reagent A and B are mixed, polymerase-fluorescein-template-primercomplex binds dCnP-Cy3 and quenches fluorescence. Mg²⁺ enables thepolymerase to incorporate the nucleotide, which releases the cleavageproduct, pyrophosphate with attached Cy3, nP-Cy3. Since the quencher isreleased, fluorescence increases. The assay can be performed with eithera stop flow device (Kintek Corp) or a fluorescent plate reader.

See FIG. 2 for a schematic representation of the assay and a graph of anexemplary reaction. See FIG. 9 for representative data.

Example 7 Attachment to Nanopore

This example provides methods of attaching a variant polymerase to ananopore, e.g., α-hemolysin.

The polymerase may be coupled to the nanopore by any suitable means.See, for example, PCT/US2013/068967 (published as WO2014/074727; GeniaTechnologies, Inc.), PCT/US2005/009702 (published as WO2006/028508;President and Fellows of Harvard College), and PCT/US2011/065640(published as WO2012/083249; Columbia University).

The polymerase, e.g., a variant pol6 DNA Polymerase, was coupled to aprotein nanopore (e.g. alpha-hemolysin), through a linker molecule.Specifically, the SpyTag and SpyCatcher system, that spontaneously formscovalent isopeptide linkages under physiological conditions was used.See, for example, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.

The pol6 variant SpyCatcher HisTag was expressed according to Example 2and purified using a cobalt affinity column. The SpyCatcher polymeraseand the SpyTag oligomerized nanopore protein were incubated overnight at4° C. in 3 mM SrCl₂. The 1:6-polymerase-template complex was thenpurified using size-exclusion chromatography.

The linker was attached at either the N-terminal or C-terminal of thepol6 variant. The N-terminally attached variants were found to be morerobust, e.g., more stable. Therefore, N-terminally attached linkers wereused.

Example 8 Activity on a Biochip

This example demonstrates the ability of a nanopore-bound variantpolymerase to bind tagged nucleotides and thereby allow for thedetection of blocked channel currents at the nanopore to which thepolymerase is attached.

The polymerase was attached to a nanopore and embedded in a lipidbilayer over a well on a semiconductor sensor chip, also called abiochip. The lipid bilayer was formed and the nanopore with attachedpolymerase was inserted as described in PCT/US2014/061853 (entitled“Methods for Forming Lipid Bilayers on Biochips” and filed 22 Oct.2014).

Variant polymerases were complexed with template DNA under low saltconditions.

The capability of the nanopore bound-variant polymerase to bind taggednucleotides was determined in static capture experiments whereby taggednucleotides are bound by the polymerase, and blocked channel current ismeasured as the tagged nucleotide is presented to the nanopore. Staticcapture experiments are performed in the presence of Ca2+, whichprevents catalysis and elongation of DNA, and allows for the detectionof repeated capture of the same type of tagged nucleotide. In thisexperiment, the tagged nucleotide used was dTnP-tag.

An exemplary polymerase variant Pol6 (S366A+N535L+I652Q) coupled to analpha hemolysin nanopore on the biochip, and was complexed with templateDNA.

The static capture of tagged thymidine nucleotide (Tag is T30 (SEQ IDNO:9)) by the Pol6 (S366A+N535L+I652Q)-DNA complex was recorded at 100mV in the presence of 20 mM Hepes7.5, 300 mM NaCl, 3 mM CaCl₂ and 5 mMTCEP above and below the bilayer.

The results are shown in FIGS. 7 and 8. The traces in FIG. 7 showelectrolytic current measured at 100 mV through the pore as a functionof time. The open pore current at this voltage was about 1 nA (uppermosttrace); and the blocked pore current at the same voltage was about 0.33nA (middle trace). The open channel current was normalized to 1according to the system software, and the blocked channel current wasdecreased by the dTnP-T30 (SEQ ID NO:9) to 33% of the open channelcurrent. The current blockades shown in this trace are associated withthe binding of thymidine polyphosphate by the variant Pol6-DNA complex,occurring in proximity to the nanopore. The corresponding uppermosthistogram in FIG. 7 (right) shows the frequency of current blockadesobserved at 100 mV with a change in current normalized to the open porecurrent in the same pore; and the histogram (right) corresponding to themiddle trace, shows the frequency of current blockades observed at 100mV with a change in current normalized to the blocked pore current inthe same pore.

FIG. 8 shows Dwell time for the static capture of tagged thymidine shownin FIG. 7. FIG. 8 (left) shows a histogram of the number of occurrencesthat tagged dTNP was bound by variant Pol6 as a function of the currentas normalized to open channel current. The average dwell time of eachcapture of dTNP-tagged nucleotide was determined to be 1.2 seconds. Thebackground capture (i.e., non-polymerase mediated) of the tag in thepore has a dwell time in the range of a few milliseconds (data notshown). A goal in the enzyme evolution was to improve the dissociationrate of the tagged polyphosphate nucleotide, so you can see dwell timeslong enough to record a polymerase mediated capture that is welldistinguished from background. As shown in FIG. 8, the average dwelltime of 1.2 sec is well above the background. The Cell index is acolor-based scheme for the approximately 8000 cells present on the chipused in this experiment.

The data show that the exemplary variant polymerase, Pol6(S366A+N535L+I652Q) is capable of binding tagged nucleotides and toallow for detection in the change in current through the nanopore towhich the polymerase is attached.

The results provide evidence that variant polymerases attached tonanopores on a biochip can bind tagged nucleotides with high fidelity,and present the tagged nucleotides to the nanopores for dwell times thatprovide sufficient time for the detection of nucleotide incorporation,and possibly for decreasing the probability of sequencing errors, e.g.,insertions, deletions, etc., during nanopore sequencing.

Example 9 Rolling Circle Amplification Assay

This example describes the amplification of a polynucleotide template ina rolling circle-based assay.

The template used was an in house template HFcirc10. It's a simplecircular template ˜150 bp long.

The assay was run in a total reaction volume of 40 μl (28 μl of ReagentA+2 μl of 2 μM Polymerase+10 μl of Reagent B).

Reagent A:

Kglu 75 mM HEPES 7.5 25 mM EDTA 0.2 mM Triton X-100 0.05 % TCEP 5 mM BSA25 μg/ml Primed Circular 100 nM template dNTPs/dN6Ps/Tags 25 μM

Reagent B:

HEPES 7.5 25 mM Kglu Varied mM Triton X-100 0.05 % TCEP 5 mM BSA 25μg/ml MgCl2 40 mM

Two μl of 2 μM polymerase were added to 28 μl Reagent A to give 1:1molar ratio of DNA to polymerase (100 nM each) in the final 40 μl assaymix. The Reagent A/polymerase mix was incubated for 10 min in this 75 mMsalt condition to allow polymerase to bind DNA.

Next, 10 μl of Reagent B were added to the to the Reagent A/polymerasemixture to start the reaction.

At pre-determined time points, 10 μl samples were removed from thereaction and added to 10 μl formamide with 50 mM EDTA to quench thereaction. Samples were taken at time points 0 min, 10 min, 30 min and 40minutes.

The formamide samples were heated to 94° C. for approximately 3 min todenature proteins and secondary structures of DNA. The samples were notallowed to cool down to 4° C. Add 2 μl of 100×SYBR GREEN or GOLD dye.

15 μl of each sample was run on a 1.2% Agarose gel for 1 hour 15 minutesat 100V.

An image of the gel was acquired using the blue tray for the Biorad GELDOC EZ imager.

FIG. 11 shows the 40 minute time point results of the assay. A molecularladder is shown in lanes 1 and 19, numbering left to right. Lane 2 has asample from t=0; no product is visible. Each of lanes 3-18 is adifferent variant pol6 polymerase.

All variants shown are able to do strand displacement and generate longkilo base DNA products with all hexaphosphate nucleotides.

Example 10 Sequencing Template DNA Using Tagged Nucleotides

This example demonstrates that the variant polymerase is functional in asequencing by synthesis method on a biochip.

AC sequencing of a heteropolymer template using Pol6-26i-D44A polymeraseat 20 mM Hepes, pH 8, 500 mM Potassium Glutamate and 3 mM MgCl2 at roomtemperature. A nanopore with attached polymerase was embedded in thelipid bilayer as described herein. Primed DNA was added and allowed tocomplex with the polymerase. Four different tagged nucleotides wereadded at a concentration of 25 μM. The sequencing by synthesis mayproceed as described in WO 2014/074727 entitled “Nucleic Acid SequencingUsing Tags.” The trace in FIG. 12 shows a sequencing accuracy of 76% fora heteropolymer template and 96 bp read length.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTING FREE TEXTSEQ ID NO: 1 - Wild-type Pol6 (DNA polymerase [Clostridium phagephiCPV4]; GenBank: AFH27113.1)   1mdkhtqyvke hsfnydeykk anfdkiecli fdtesctnye ndntgarvyg wglgvtrnhn 061miygqnlnqf wevcqnifnd wyhdnkhtik itktkkgfpk rkyikfpiav hnlgwdvefl 121kyslvengfn ydkgllktvf skgapyqtvt dveepktfhi vqnnnivygc nvymdkffev 181enkdgsttei glcldffdsy kiitcaesqf hnyvhdvdpm fykmgeeydy dtwrspthkq 241ttlelryqyn diymlrevie qfyidglcgg elpltgmrta ssiafnvlkk mtfgeektee 301gyinyfeldk ktkfeflrkr iemesytggy thanhkavgk tinkigcsld inssypsqma 361ykvfpygkpv rktwgrkpkt eknevyliev gfdfvepkhe eyaldifkig avnskalspi 421tgavsgqeyf ctnikdgkai pvykelkdtk lttnynvvlt sveyefwikh fnfgvfkkde 481ydcfevdnle ftglkigsil yykaekgkfk pyvdhftkmk venkklgnkp ltnqakliln 541gaygkfgtkq nkeekdlimd knglltftgs vteyegkefy rpyasfvtay grlqlwnaii 601yavgvenfly cdtdsiycnr evnsliedmn aigetidkti lgkwdvehvf dkfkvlgqkk 661ymyhdckedk tdlkccglps darkiiigqg fdefylgknv egkkqrkkvi ggcllldtlf 721tikkimf SEQ ID NO: 2 - Pol6 (with His tag)MHHHHHHHHS GGSDKHTQYV KEHSFNYDEY KKANFDKIEC LIFDTESCTN   50YENDNTGARV YGWGLGVTRN HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT  100IKITKTKKGF PKRKYIKFPI AVHNLGWDVE FLKYSLVENG FNYDKGLLKT  150VFSKGAPYQT VTDVEEPKTF HIVQNNNIVY GCNVYMDKFF EVENKDGSTT  200EIGLCLDFFD SYKIITCAES QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH  250KQTTLELRYQ YNDIYMLREV IEQFYIDGLC GGELPLTGMR TASSIAFNVL  300KKMTFGEEKT EEGYINYFEL DKKTKFEFLR KRIEMESYTG GYTHANHKAV  350GKTINKIGCS LDINSSYPSQ MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI  400EVGFDFVEPK HEEYALDIFK IGAVNSKALS PITGAVSGQE YFCTNIKDGK  450AIPVYKELKD TKLTTNYNVV LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN  500LEFTGLKIGS ILYYKAEKGK FKPYVDHFTK MKVENKKLGN KPLTNQAKLI  550LNGAYGKFGT KQNKEEKDLI MDKNGLLTFT GSVTEYEGKE FYRPYASFVT  600AYGRLQLWNA IIYAVGVENF LYCDTDSIYC NREVNSLIED MNAIGETIDK  650TILGKWDVEH VFDKFKVLGQ KKYMYHDCKE DKTDLKCCGL PSDARKIIIG  700QGFDEFYLGK NVEGKKQRKK VIGGCLLLDT LFTIKKIMF*  739SEQ ID NO: 3 - Pol6 with His-tag (DNA sequence) ATGCATCACC ATCATCATCA CCACCAC AGC GGCGGTTCCG ACAAACACAC   50GCAGTACGTC AAAGAGCATA GCTTCAATTA TGACGAGTAT AAGAAAGCGA  100ATTTCGACAA GATCGAGTGC CTGATCTTTG ACACCGAGAG CTGCACGAAT  150TATGAGAACG ATAATACCGG TGCACGTGTT TACGGTTGGG GTCTTGGCGT  200CACCCGCAAC CACAATATGA TCTACGGCCA AAATCTGAAT CAGTTTTGGG  250AAGTATGCCA GAACATTTTC AATGATTGGT ATCACGACAA CAAACATACC  300ATTAAGATTA CCAAGACCAA GAAAGGCTTC CCGAAACGTA AGTACATTAA  350GTTTCCGATT GCAGTTCACA ATTTGGGCTG GGATGTTGAA TTCCTGAAGT  400ATAGCCTGGT GGAGAATGGT TTCAATTACG ACAAGGGTCT GCTGAAAACT  450GTTTTTAGCA AGGGTGCGCC GTACCAAACC GTGACCGATG TTGAGGAACC  500GAAAACGTTC CATATCGTCC AGAATAACAA CATCGTTTAT GGTTGTAACG  550TGTATATGGA CAAATTCTTT GAGGTCGAGA ACAAAGACGG CTCTACCACC  600GAGATTGGCC TGTGCTTGGA TTTCTTCGAT AGCTATAAGA TCATCACGTG  650TGCTGAGAGC CAGTTCCACA ATTACGTTCA TGATGTGGAT CCAATGTTCT  700ACAAAATGGG TGAAGAGTAT GATTACGATA CTTGGCGTAG CCCGACGCAC  750AAGCAGACCA CCCTGGAGCT GCGCTACCAA TACAATGATA TCTATATGCT  800GCGTGAAGTC ATCGAACAGT TTTACATTGA CGGTTTATGT GGCGGCGAGC  850TGCCGCTGAC CGGCATGCGC ACCGCTTCCA GCATTGCGTT CAACGTGCTG  900AAAAAGATGA CCTTTGGTGA GGAAAAGACG GAAGAGGGCT ACATCAACTA  950TTTTGAATTG GACAAGAAAA CCAAATTCGA GTTTCTGCGT AAGCGCATTG 1000AAATGGAATC GTACACCGGT GGCTATACGC ACGCAAATCA CAAAGCCGTT 1050CCTAAGACTA TTAACAAGAT CGGTTGCTCT TTGGACATTA ACAGCTCATA 1100CCCTTCGCAG ATGGCGTACA AGGTCTTTCC GTATGGCAAA CCGGTTCGTA 1150AGACCTGGGG TCGTAAACCA AAGACCGAGA AGAACGAAGT TTATCTGATT 1200GAAGTTGGCT TTGACTTCGT GGAGCCGAAA CACGAAGAAT ACGCGCTGGA 1250TATCTTTAAG ATTGGTGCGG TGAACTCTAA AGCGCTGAGC CCGATCACCG 1300GCGCTGTCAG CGGTCAAGAG TATTTCTGTA CGAACATTAA AGACGGCAAA 1350GCAATCCCGG TTTACAAAGA ACTGAAGGAC ACCAAATTGA CCACTAACTA 1400CAATGTCGTG CTGACCAGCG TGGAGTACGA GTTCTGGATC AAACACTTCA 1450ATTTTGGTGT GTTTAAGAAA GACGAGTACG ACTGTTTCGA AGTTGACAAT 1500CTGGAGTTTA CGGGTCTGAA GATTGGTTCC ATTCTGTACT ACAAGGCAGA 1550GAAAGGCAAG TTTAAACCTT ACGTGGATCA CTTCACGAAA ATGAAAGTGG 1600AGAACAAGAA ACTGGGTAAT AAGCCGCTGA CGAATCAGGC AAAGCTGATT 1650CTGAACGGTG CGTACGGCAA ATTCGGCACC AAACAAAACA AAGAAGAGAA 1700AGATTTGATC ATGGATAAGA ACGGTTTGCT GACCTTCACG GGTAGCGTCA 1750CGGAATACGA GGGTAAAGAA TTCTATCGTC CGTATGCGAG CTTCGTTACT 1800GCCTATGGTC GCCTGCAACT GTGGAACGCG ATTATCTACG CGGTTGGTGT 1850GGAGAATTTT CTGTACTGCG ACACCGACAG CATCTATTGT AACCGTGAAG 1900TTAACAGCCT CATTGAGGAT ATGAACGCCA TTGGTGAAAC CATCGATAAA 1950ACGATTCTGG GTAAATGGGA CGTGGAGCAT GTCTTTGATA AGTTTAAGGT 2000CCTGGGCCAG AAGAAGTACA TGTATCATGA TTGCAAAGAA GATAAAACGG 2050ACCTGAAGTG TTGCGGTCTG CCGAGCGATG CCCGTAAGAT TATCATTGGT 2100CAAGGTTTCG ACGAGTTTTA TCTGGGCAAA AATGTCGAAG GTAAGAAGCA 2150ACGCAAAAAA GTGATCGGCG GTTGCCTGCT GCTGGACACC CTGTTTACGA 2200TCAAGAAAAT CATGTTCTAA 2220

CITATION LIST Patent Literature

-   [1] PCT/US2005/009702 (published as WO2006/028508 on 16 Mar. 2006;    President and Fellows of Harvard College; entitled METHODS AND    APPARATUS FOR CHARACTERIZING POLYNUCLEOTIDES).-   [2] PCT/US2011/065640 (published as WO2012/083249 on 21 Jun. 2012;    Columbia University; entitled DNA SEQUENCING BY SYNTHESIS USING    MODIFIED NUCLEOTIDES AND NANOPORE DETECTION).-   [3] PCT/US2013/068967 (published as WO2014/074727 on 15 May 2014;    Genia Technologies; entitled NUCLEIC ACID SEQUENCING USING TAGS).-   [4] PCT/US2013/046012 (Genia Technologies, Inc., entitled CHIP    SET-UP AND HIGH-ACCURACY NUCLEIC ACID SEQUENCING, published 19 Dec.    2013 as WO2013/188841).-   [5] US 2013/0053544 (Isis Innovation Limited) entitled Peptide Tag    Systems That Spontaneously Form an Irreversible Link to Protein    Partners via Isopeptide Bonds, published 28 Feb. 2013.

Non-Patent Literature

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What is claimed is:
 1. A modified deoxyribonucleic acid (DNA) polymerasehaving a DNA polymerase activity, said modified DNA polymerasecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:2, which amino acid sequence includes an amino acidsubstitution at position N224, wherein the substitution is selected fromthe group consisting of N224Q, N224M, N224R, and N224K.
 2. The modifiedDNA polymerase of claim 1, wherein modified DNA polymerase is capable ofincorporating a tagged deoxyribonucleotide tetraphosphate, a taggeddeoxyribonucleotide pentaphosphate, a tagged deoxyribonucleotidehexaphosphate, a tagged deoxyribonucleotide heptaphosphate, and/or atagged deoxyribonucleotide octophosphate into a growing DNA strand. 3.The modified DNA polymerase of claim 2, wherein the tag is attached to aterminal phosphate of the polyphosphate deoxyribonucleotide.
 4. Themodified DNA polymerase of claim 1, wherein the modified DNA polymerasehas an altered characteristic as compared to a DNA polymerase consistingof SEQ ID NO:2.
 5. The modified DNA polymerase of claim 1, wherein theamino acid sequence further includes T529M, S366A, A547F, N545L, Y225L,and D657R substitutions.
 6. The modified DNA polymerase of claim 1,wherein the substitution at position N224 is N224R.
 7. A compositioncomprising the modified DNA polymerase of claim 1 attached to ananopore.
 8. The composition of claim 7, wherein the nanopore is analpha-hemolysin nanopore comprising 7 alpha-hemolysin monomers.
 9. Thecomposition of claim 8, wherein 1 of the alpha-hemolysin monomers isattached to the modified DNA polymerase and 6 of the alpha-hemolysinmonomers are not attached to a DNA polymerase.
 10. The composition ofclaim 9, wherein the alpha-hemolysin nanopore is inserted into amembrane adjacent to a sensing electrode, such that the sensingelectrode is capable of detecting a current flowing through thenanopore.
 11. The composition of claim 10, wherein the membrane is alipid bilayer.
 12. The composition of claim 7, wherein the amino acidsequence further includes T529M, S366A, A547F, N545L, Y225L, and D657Rsubstitutions.
 13. The composition of claim 12, wherein the substitutionat position N224 is N224R.
 14. A method of sequencing a targetdeoxyribonucleic acid (DNA) molecule, the method comprising: (a) bindingthe target DNA molecule to the modified DNA polymerase of claim 1,wherein the modified DNA polymerase is attached to a nanopore; (b)incorporating a tagged polyphosphate deoxyribonucleotide into a growingDNA strand by a template-dependent polymerization catalyzed by themodified DNA polymerase, wherein a tag of the tagged polyphosphatedeoxyribonucleotide is inserted into the nanopore during thepolymerization, (c) detecting a change in a current flowing through thenanopore caused by insertion of the tag into the nanopore; and (d)correlating the change in the current to the identity of the taggedpolyphosphate deoxyribonucleotide incorporated into the growing DNAstrand, thereby sequencing the target DNA molecule.
 15. The method ofclaim 14, wherein the nanopore is an alpha-hemolysin heptamer.
 16. Themethod of claim 15, wherein the polyphosphate deoxyribonucleotide isselected from the group consisting of a tetraphosphate, apentaphosphate, a hexaphosphate, a heptaphosphate, and an octophosphate.17. The method of claim 14, wherein the tag is attached to a phosphateof the polyphosphate deoxyribonucleotide.
 18. The method of claim 14,wherein the tag is attached to a terminal phosphate of the polyphosphatedeoxyribonucleotide.
 19. The method of claim 14, wherein the amino acidsequence further includes T529M, S366A, A547F, N545L, Y225L, and D657Rsubstitutions.
 20. The method of claim 19, wherein the substitution atposition N224 is N224R.